18
DNA is Profligate …sticky ends with complementary base pairs can form hydrogen bonds, …with DNA from any species.

19
Requires a Vector…...a specialized DNA sequence that can enter a living cell,...signal its presence to an investigator,... and provide a means of replication for itself and the foreign DNA it carries. Cloning …specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study.

20
And, Vectors… …contain unique restriction sites to facilitate the creation of recombinant DNA molecules,... must also possess a distinguishing physical characteristic such as size or shape by which it can be purified away from the host cells genome.

25
Amplification …if you were to grind me up with a giant mortar and pestle, and extract all of my DNA coding for a single gene, you’d get about 1  g of DNA, …two liters of bacterial culture carrying my gene in a recombinant DNA vector, would yield an equivalent mass of DNA.

26
Assignment Read 10.1 and be prepared to clone a fragment of DNA using a plasmid as a vector.

36
Denaturing can’t use helicase in vitro …DNA denaturing conditions such as high heat or low salt concentrations irreversibly denature or inactivate most polymerases, …dNTPs are not affected by denaturation, …primers are not affected by denaturation.

37
Making Two More Strands Add polymerase, etc. add primer to second strand

38
Denaturation Step Bad …several rounds of in vitro replication could be performed (prior to PCR), however, accumulation of denatured polymerases quickly poisoned the reactions.

39
…bacteria discovered in a hot spring in Yellowstone Natural Park in 1965, …lives in salty water that ranges from 70 o - 75 o C, …thus, does DNA replication at very high temperatures.

49
PCR Applications …new applications are created every day, PCR products can be used for mapping genes, PCR products can be used as probes, PCR products can be probed, PCR can be used to identify genotypes, PCR can be used to sequence DNA directly.

50
Molecular Probing Heterologous Hybridization …genes (DNA), or gene products (RNA) can be identified based on hybridization to labeled molecules, …DNA probes are short, single-stranded stretches of nucleic acid that are complementary to target nucleic acids, – s of base pairs in length, …radioactive or fluorescent labeled for detection.

74
Cycle Sequencing Chain Termination Template ddNTPsdNTPs Lots of each sized fragment are produced, each with a specific florescent base on the end.base etc. Another Template

75
Dosage Compensation …more Chapter 7 X chromosomes in females provide twice the genes, as in males, –Drosophila: female genes are expressed at 50% of the male levels, –Mammals: one X ho,olog in females is silenced.

76
Canadian Cat Scientists Sees it First Barr Body

77
Lyon Hypothesis Mary Lyon; in humans, X chromosomes from father and mother are randomly inactivated.

78
X Inactivation The structure of the chromosome is altered. Barr Body

79
X-Linked Mosaicism Different cell lineages contribute to different body locations on the body.

80
Epigenesis A change in gene regulation brought about without a change in DNA sequence, –often to the structure of the chromosome, –or through modification of the nucleotide bases, –or through post transcriptional regulation.